Francisella tularensis is a facultative intracellular pathogen of macrophages and the causative agent of tularemia. Although it is clear that inhalation of as few as ten F. tularensis subsp. tularensis can be fatal, and that macrophages are the major reservoir of this organism in vivo, how Ft evades elimination is only beginning to be defined. Within a few hours of uptake, Ft breaches the phagosome membrane and replicates in the cytosol. Our recent data demonstrate for the first time that fully virulent Ft strain SchuS4 disrupts phagosome maturation at an earlier stage of the pathway than has previously been appreciated and escapes from a novel compartment that excludes the late endoosome markers Rab7, mannose-6-phosphate receptor, lysobisphosphatidic acid and cathepsin S, despite local accumulation of lamp-1. Generation of this compartment requires live, MglA-positive Ft and is achieved via the ability of this pathogen to inhibit profoundly the activity of protein kinase C-a (PKCa) throughout infected cells. Thus, our data identify PKCa as the first host factor known to be targeted by Ft to evade intracellular killing. PKCa is also required for other aspects of innate defense, and our preliminary data support the hypothesis that MHC class II antigen presentation and oxidative defense mechanisms may also be impaired. At the same time, how Ft breaches the phagosome membrane is unclear, and we present evidence to support a model in which clathrin adapters and Rab family GTPases play important roles in phagosome dissolution and bacterial escape to the cytosol. From the bacterial perspective, few Francisella virulence factors have been identified and their mechanisms of action remain obscure. We recently developed a Tn5 transposon mutagenesis system for Francisella that we have used to identify mutants in FTL0347 and FTL1542 which encode a hypothetical membrane permease and acyl-CoA synthetase, respectively. Expression of known virulence factors is markedly reduced in both these mutants and preliminary characterization suggests that these genes are required for phagosome escape and inhibition of the phagocyte respiratory burst, respectively. Additionally, we constructed a TraSH (Transposon Site (Hybridization) derivative of our transposon that will allow us to identify novel genes required for Ft entry, growth and survival in human macrophages, Accordingly, our Specific Aims are: 1) to elucidate the mechanisms and functional consequences of Ft-mediated inhibition of PKCa signaling in macrophages; and 2) to identify and characterize novel F. tularensis genes required for evasion of innate macrophage defenses using transposon mutagenesis, including TRASH.